Use of isolated domains of type IV collagen to modify cell and tissue interactions

ABSTRACT

The instant invention demonstrates that the 7S domain of type IV collagen disrupts cell aggregation and tissue development. Structural changes in mesoglea, inhibition of cell proliferation, and changes in cell differentiation patterns accompanies the blockage of cell aggregates which indicate that blockage may be due to alterations in mesoglea (extracellular matrix) structure with accompanying effects on cell behavior. Type IV collagen has a critical role in the initial formation of mesoglea and that perturbation of mesoglea formation affects cell division, cell differentiation, and morphogensis.

STATEMENT OF RIGHTS

[0001] The U.S. Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms ofGrants No. 01-RR06500 and AM 18381 awarded by the National Institute ofHealth.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods for the manipulation ofintercellular and intertissue interactions. The instant inventionprovides methods for the inhibition of cell adhesion to extracellularmatrix components or the formation of functional basal lamina, and themanipulation of the results of such attachments. Thus the instantinvention to the modification of cellular interactions, withextracellular components, and methods for maintaining cell phenotype,developmental stage, and plasticity in vivo and in vitro.

[0004] 2. Description of the Prior Art

[0005] The basement membrane (basal lamina) is a sheet-likeextracellular matrix which is a basic component of all tissues. Thebasal lamina provides for the compartmentalization of tissues, and actsas a filter for substances traveling between tissue compartments.Typically, the basal lamina is found closely associated with anepithelium, or endothelium in all tissues of an animal including bloodvessels and capillaries. The basal lamina components are secreted bycells, and then self assemble to form an intricate extracellularnetwork. The formation of a biologically active basal lamina isimportant to the development and differentiation of the associatedcells.

[0006] The Cnidarian, Hydra, is a simplified metazoan whose body wall iscomposed of an epithelial bilayer with an intervening extracellularmatrix (ECM) termed the mesoglea. Hydra mesoglea have been shown to havea number of components seen in the ECM or basement membranes of higherinvertebrates and vertebrates, these include: fibronectin, type IVcollagen, laminin, and heparan sulfate proteoglycan. Hydra cellaggregation involves the complete morphogensis of adult hydra frompellets of dissociated hydra cells. During this developmental process,cells segregate into an epithelial bilayer and then deposit a newextracellular matrix prior to the continuation of morphogensis.

[0007] Extracellular matrix (ECM) components play a critical role indevelopment through their affects on such cell processes as celldivision, cell attachment, cell migration, and cell differentiation(reviewed by Timpl et al., 1989, Int. Rev. Exp. Pathol. 29:1-112; Damskyand Bernfield. 1991, Current Opn. in Cell Bio. 3:777-778;. Hynes, 1992,Cell 69:11-25). It has been established that ECM/cell interactions areutilized by a wide range of vertebrate and invertebrate species toinclude such primitive organisms as the Cnidarian, Hydra. Hydra isparticularly interesting in this regard because it represents one of thefirst animal phyla to develop defined tissue layers separated by anacellular extracellular matrix (Field et al., 1988, Science239:748-752). Previous studies have shown that hydra ECM, termedmesoglea, contains type IV collagen, laminin, fibronectin, and heparansulfate proteoglycans (Sarras et al., 1991a. Dev. Biol. 148:481-494).These molecules are continuously synthesized and deposited into themesoglea in adult hydra and during hydra head regeneration (Hausman etal., 1971. J. Exp. Zool. 177:435-446). Other studies have shown thatdevelopmental processes in hydra such as head regeneration are dependenton the normal formation of ECM. These studies have shown that headregeneration in hydra morphogensis can be blocked by using drugs thatperturb collagen cross linking or drugs that interfere with proteoglycanGAG chain extension (Sarras et al., 1991b, Dev. Biol. 148:495-500).These studies have most recently been extended to the hydra cellaggregate system. This system allows one to form a pellet withdissociated hydra cells and then observe the complete regeneration ofthe adult hydra body within 72-96 hours though the process ofcytodifferentiation and morphogensis (Gierer et al., 1972, Nature NewBiol. 239:98-101; Sarras et a, 1993, Dev. Biol. 157:383-398). Suchstudies of hydra development and the role of the ECM have focusedheavily on a chemical approach (Barzanski et al., 1974, Amer. Zool.14:575-581; Sarras et al., 1991ab, supra). Hydra cell aggregates firstform an epithelial bilayer and then deposit an ECM before morphogensisproceeds. Hydra cell aggregate development is blocked by drugs thatperturb ECM formation and by antibodies raised against isolated hydramesoglea. These studies demonstrate that functional studies of ECM/cellinteraction can be carried out under in vivo conditions with hydra.

[0008] Type IV collagen has been shown to be a major structuralcomponent of basement membranes and has also been shown to be present inhydra ECM. The protomeric form of type IV collagen is formed as aheterotrimer made up from a number of different subunit chains calledα1(IV), α2(IV) etc. The type IV collagen heterotrimer is characterizedby three distinct structural domains: the non-collagenous (NC1) domainat the carboxyl terminus: the triple helical collagenous domain in themiddle region; and the 7S collagenous domain at the amino terminus(FIG. 1) (Martin et al., 1988, Adv. Protein Chem. 39:1-50; Gunwar etal., 1991, J. Biol. Chem. 266:14088-14094). Type IV collagen exists as asupramolecular structure in ECM and this structure is thought to serveas a framework which provides mechanical stability to ECM (Timpl et al.,1986, supra) and as a scaffolding for the binding and alignment of otherECM molecules such as fibronectin, laminin, eutecin, and heparan sulfateproteoglycans (Gunwar et al., 1991, supra). The biological function oftype IV collagen is critically related to the formation of an intact ECMsince disruption of collagen cross linking by β-aminopropionitrileinterferes with the mesoglea formation and this leads to a blockage innormal hydra morphogensis (Sarras et al., 1991b, 1993, supra).

[0009] Hydra cell aggregate development involves complete morphogensisof adult hydra structures within 96 hr from pellets formed withdissociated hydra cells (Grierer et al., 1972, supra; Sato et al. 1992,Dev. Biol 151:111-116; Technau et al., 1992, Dev. Biol. 151: 117-127:Sarras et al., 1993, supra ). Morphologically, hydra cell aggregatedevelopment can be divided into two stages. The initial stage is fromTime 0 to 24 hr when aggregates develop from a solid cell mass into afluid-filled cyst where the outer wall is formed from an epithelialbilayer with an intervening ECM termed mesoglea. This stage involvesactive cell sorting between ectodermal and endodermal cells (Technau etal., 1992, supra) and subsequent mesoglea formation once the bilayer isestablished. The later developmental stages (24-96 hr) involve processesnormally associated with tissue histogenesis; namely, alterations in theshape of epithelial layers, cell migration, cell differentiation, andother processes that result in morphogensis of foot, head, and tentaclestructures. In regard to the initial stages of hydra cell aggregatedevelopment, it has been shown that head regeneration in aggregates isnot due to the clustering of cells from the original head regions. Ithas been suggested that head regeneration arises de novo (Gierer et al.,1972. supra ; Technau et al., 1992, supra) from foci of developmentgradients established around the spherical aggregate. This indicatesthat cell differentiation or transdifferentiation into head region cellsactively occurs during hydra cell aggregate development. In addition topositional information and possible activator influences, cells maydifferentiate or transdifferentiate under the influences of otherdevelopmental cues such as signals arising from the ECM.

[0010] Previous studies have shown that in vertebrates, fibronectininteracts with various collagens during matrix assembly, including typeIV collagen (Carter, 1984, J. Cell Bio. 99:105-114). In addition,antibodies to the collagen binding domain of fibronectin had the abilityto block ECM assembly by human lung fibroblasts. (McDonald, 1982, J.Cell Biol. 92:485-492). Other studies raised doubts as to theinteraction, while polyclonal antibodies to the collagen binding domainblocked matrix assembly, purified collagen binding domains had noinhibitory effects in this assembly process (McDonald et al., 1987, J.Biol. Chem. 262:2957-2967; Hynes, 1990, Cell 48:549-554). In generalfibronectin (FN) appears before collagen during assembly of vertebratematrices, however, in the case of hydra ECM formation, FN and collagenappear in the mesoglea about the same time, based on immunofluorescentstudies. Type IV collagen has been implicated as ant in several humandiseases (Hudson et al., 1993, J. Biol. Chem. 268:26033-26036). Basementmembrane and its components have a role in lymphocyte adhesion,migration and proliferation (Li and Cheung, 1992, J. of Immunology149:3174-3181).

[0011] The fundamental role ECM plays in tissue development and celldifferentiation reverberates across phyla and kingdoms, to focusattention on the most basic elements that are required for all tissueinteractions. The use of hydra as a model system for the study of basicelements of complex tissue interactions is a recognized approach.Instead of attempting to deduce the interaction between isolated tissuesof higher order animals, the same mechanisms and phenomenon can beexamined in vivo by using the complete animal, in hydra. This approachhas led to the use of hydra to study the effects of glucose on tissuemorphology, in an effort to understand the pathological effects ofuncontrolled diabetes on kidney glomeruli, with excellent results (Zhanget al., 1990, Diabetologia 33:704-707).

[0012] Recently the β1-laminin gene has been cloned and sequenced inhydra, showing very high homology with the human counter part. Thehomologues of fibronectin and collagen are present as well. It is areflection on the fundamental role ECM plays, that hydra and higherorder animals show the same cell matrix interactions, with similarcomponents, domain interactions, receptor molecules and response toextracellular signals. Mammalian and even human hormones, when appliedto hydra result in bioactivity and effect on cell behavior. It ispossible to use human insulin to stimulate cell proliferation in hydra.Other such cross-phyla activities can be attributed to many growthfactors as well, i.e. EGF (epidermal growth factor), TGF-β, FGF(fibroblast growth factor), PDGF, to name a few.

[0013] Specific methods for the manipulation of cell adhesion to ECM,basal lamina, or adjacent cells would be useful for the in vivomanipulation of tissues and cells. Methods which address the fundamentalelements of basic cell and tissue interactions are applicable to allsystems which exhibit similar characteristic features. Such in vivo usesinclude, and are not limited to, inhibition of basal lamina formation,inhibition of basal lamina/cell interactions, and to encourage cells tomaintain phenotypic plasticity. Such methods will also be useful for thein vitro manipulation of cells and tissues, for instance in maintainingcell cultures in undifferentiated or homeostatic states, non-enzymaticdispersal of cells from attachments, or the maintenance of confluentcells in suspension for propagation, maintenance, or collection.

SUMMARY OF THE INVENTION

[0014] The instant invention provides methods for inhibiting basallamina membrane formation, in cell or tissue development, comprisingcontacting the cell or tissue with one or more isolated domains of typeIV collagen. The instant invention also provides methods for in vitrocultivation of cells comprising contacting the cells to be cultivatedwith an isolated domain of type IV collagen to disrupt the formation ofbasal lamina extracellular matrix contacts. The instant inventionfurther provides methods for disrupting basal lamina type IV collagen.In a specific embodiment of the instant invention, the isolated domainof type IV collages is the 7S or NC1 domain, or protein constructshaving substantially the same structure as the active elements withinthe 7S or NC1 domain.

[0015] Thus the instant invention provides methods for the interferencewith cell interactions with basal lamina components which comprisescontacting the cells or tissues with an isolated domain of type IVcollagen, and in a preferred embodiment the isolated domain is eitherthe 7S domain or the NC1 domain of type IV collagen or substantiallyhomologous protein constructs thereof which contain the specificstructural elements within the 7S and NC1 domain that convey activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 are two diagrams illustrating segments of type IV collagen(1A) and fibronectin proteolytic fragments (1B) used in blockingexperiments.

[0017]FIG. 2 is a graph which illustrates results of the blockage ofaggregate morphogenesis observed with NC1 domain monomer and 7S domain,when all type IV collagen fragments are compared on an equal molar basis(0.03 μM). From left to right: control group; NC1 monomer, NC1 hexamer,8-kDa fragment of NC1; 7S domain; type I collagen. Results are reportedas % of normal morphogenesis as described below.

[0018]FIG. 3 are photographs of representative Hydra cell aggregates at% hr of development. Control aggregates develop head and tentaclestructures (3A) while blocked aggregates remain in the characteristic24-hr cystic stage (3B). Bar=286 μm.

[0019]FIG. 4 is a graph which illustrates the concentration effect ofthe various blocking agents. At higher concentrations, NC1 domainhexamer also showed blocking effect, whereas no blockage was observedwith the 80-kDa fragment of NC1. From left to right control group; NC1monomer (MN) at 0.03 μM; NC1 domain hexamer (HEX) at 0.1 μM; NC1 domainhexamer (HEX) at 0.03 μM; NC1 domain 80-kDa fragment (80K) at 0.3 μM;NC1 domain 80-kDa fragment (80K) at 0.03 μM; 7S domain (7S) at 0.1 μM;and 7S domain (7S) at 0.03 μM. Results are reported as % of normalmorphogenesis as described below.

[0020]FIG. 5 are electron micrographs showing representative morphologyof control (5A) and blocked (5B) aggregate cells.

[0021]FIG. 6 is a graph which illustrates the results of the blockage ofaggregate formation using fibronectin and fibronectin derivedproteolytic fragments. All samples were tested at 0.5 mg/ml. From leftto right: BSA (Bovine Serum Albumin); FN (intact fibronectin); 120K (the120-kDa fragment of FN); 45K (the 45-kDa fragment of FN); 40K (the40-kDa fragment of FN); 30K (the 30-kDa fragment of FN); RGDS (RGDSpeptide); RADS (RADS peptide). Results are reported as % of normalmorphogenesis as described below. The effectiveness of the blockingagents was found to be concentration dependent.

[0022]FIG. 7 illustrates the effects of NC1 (Hexamer) and 7S domains ofType IV collagen at a 50 ug/ml concentration on angiogenesis from mousethoracic aorta organ cultures.

[0023]FIG. 8 illustrates the effects of 7S domain of Type IV collagen onangiogenesis from mouse thoracic aorta organ cultures. The domainconcentrations employed in this experiment were 0 ug/ml (control); 0.5ug/ml; 5 ug/ml and 50 ug/ml.

[0024]FIG. 9 illustrates the effects of NC1 (Hexamer) domain of Type IVcollagen on angiogenesis from mouse thoracic aorta organ cultures. Thedomain concentrations employed in this experiment were 0 ug/ml(control); 0.5 ug/ml; 5 ug/ml and 50 ug/ml.

[0025]FIG. 10 are photographs of mouse thoracic aorta segments embeddedin Matrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture. Control specimen (0ug/ml of NC1 (Hexamer) and 7S domains) exhibited growth of microvesselsfrom the cultured tissue into the matrix (FIG. 10A). In contrast,angiogenesis was inhibited in specimens cultured with 50 ug/ml of 7Sdomain (FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Our studies indicate that the formation of an appropriate ECMstructure involving fibronectin and type IV collagen is critical to cellaggregate development and that perturbation of ECM formation adverselyaffects cell division and cell differentiation during the formation ofcomplex cell aggregations and tissues. This is illustrative of thecritical role the ECM and cell-ECM interactions play in tissuedevelopment in all organisms that have differentiated tissues.Structural changes in mesoglea, inhibition of cell proliferation, andchanges in cell differentiation patterns accompanied the blockage ofcell aggregates. Thus type IV collagen is critical to the early stagesof cell aggregate development when the mesoglea is initially formed andthat perturbation of aggregate development by fragments of type IVcollagen results in alterations in hydra cell division, celldifferentiation, and morphogenesis. It is demonstrated that type IVcollagen components ace a critical element in the formation of any cellto ECM (basal lamina) contacts, and that by applying the proper methods,this interaction can be manipulated in a predictable fashion.

[0027] The following examples are meant to illustrate specificembodiments of the instant invention, and are in no way intended tolimit the breadth or scope of the teachings embodied in the instantspecification. One with ordinary skill in the art will be able to takethe teachings of the instant specification and use the instant inventionin other specific embodiments.

EXAMPLE 1 Disruption of Cell Aggregate Development

[0028] All experiments utilized Hydra vulgarie (previously named Hydraattenuata). Animals were cultured as described previously (Sarras et al.1991a, supra) and were not fed for 24 prior to use.

[0029] Hydra cell aggregates were prepared according to Gierer et al.(1972, supra) with modification described by Sans et al. (1993. supra).They were incubated either in microtiter plates (NUNS, Denmark) with oneaggregate/well/10 μg in on solution or in 96-well plates (Falcon) withthree to five aggregates/well/50 μl incubation solution. Antibioticswere used throughout all steps of aggregate preparation to assure thatthe aggregates used in this study were free of any symbionic bacterialpopulations (Sarra et al., 1993, supra). Various analysis indicated thataggregates were free of bacteria under these conditions. By 24 hr ofcell pellet formation, hydra cell aggregates develop septate junctionsbetween epithelial cells (Wood et at., 1980. J. Ultrastruc. Res.70:104-117). These junctions prevent the introduction of macromoleculesfrom medium into mesoglea. Therefore, exogenous matrix probes (e.g..fibronectin, type IV collagen, and type IV collagen fragments, peptidesor antibodies) were added to the culture medium immediately after pelletformation (Time 0). Control hydra cell aggregates were cultured in hydramedium, bovine serum albumin (Sigma. St. Louis, Mo.), or nonimmuneserum. After 24 hr of incubation, aggregates (both control andexperimental groups) were transferred into fresh hydra medium andculture was continued until 96 hr.

[0030] Type IV collagen fragments used in blocking experiments areillustrated in FIG. 1 (A). The NC1 monomer—a mixture of α1(IV), α2(IV);NC1 hexamer, 80-kDa protomeric truncated fragment (NC1 domain with partof triple helix); and the 7S domain, were all obtained from enzymaticdigestion and subsequent chromatographic purification of bovine kidneyglomeruli basement membrane or bovine lens basement membrane (Langeveldet al., 1988, J. Biol. Chem. 263: 10481-10488; Gunwar et al., 1991,supra). Antibody to the NC1 (α1(IV)+α2(IV)) domain of type IV collagenwas generated in rabbits immunized with monomeric subunits isolated fromthe globular domain of bovine kidney basement membrane type IV collagen(Langeveld et al.. 1988, supra). The precise molar concentrations ofthese fragments was determined by spectrophotometry and amino acidcomposition analysis

[0031] Criteria for morphogenesis was as follows. The morphologicaldevelopment of hydra cell aggregates was studied using a dissectingmicroscope (Wild, Herbrugg). Observations were carried out at Time 0,24, 48, 72, and 96 hr. The normal morphogenesis of hydra cell aggregatesbetween Time 0 and 96 hr has been described previously by Gierer et al.,(1972, supra) and Sums et al., (1993, supra). Abnormal development(blockage) of hydra cell aggregates was considered if an aggregate didnot show head and tentacle structures at 96 hr. i.e., was retained in acystic stage (FIG. 3). The percentage of aggregates-with head andtentacle structures at 96 hr of development was calculated and data foreach group from different experiments were pooled and plotted. A minimumof five aggregates were tested per group and all experiments wererepeated at least three times except where indicated. Using an ANOVAstatistical test, a P value <0.05 was taken as the level of significantdifference in all groups analyzed. The ANOVA Apical test was used forall data analysis.

[0032] Transmission electron microscopy was used to examine the finestructure and morphology of control and treated aggregates. Cellaggregates were immersed in Karnovsky's fixative overnight at 4° C. andpostfixed in 1% OsO₄ for 1 hr. Samples were then stained en bloc in 0.5%uranyl magnesium acetate overnight at 4° C. After dehydration andinfiltration, samples were embedded in Spurr's resin. Blocks were cutwith a Reichert-Jung microtome, stained with uranyl acetate and leadcitrate, and viewed using a JOEL 100S transmission electron microscope.Morphometry analysis was carried out as described previously (Zhang etal., 1990, supra).

[0033] Immunofluorescent screening for type IV collagen in hydra cellaggregates at various time points after pellet formation showed thatsignal was detected at 48 hr, and became progressively stronger andremained in the mesoglea throughout all later stages of aggregatedevelopment. FIG. 3 illustrates the typical appearance of control andblocked aggregates at 96 hr. Blocked aggregates failed to develop beyondthe 24 hr stage, and either remaining in a cystic stage ordisaggregating into dissociated cells by 96 hr.

[0034] As shown in FIG. 2, the 7S domain, and monomers of NC1 domainwere most effective in blocking hydra cell aggregate morphogenesis whenfragments were tested on an equal molar basis. This blockage was alsoconcentration dependent. NC1 hexamer blocked aggregate development athigher concentrations whereas the 80-kDa fragment showed no effect (FIG.4). Antibody to NC1 domain had a similar blocking effect (Table 1).TABLE 1 Treatment with Antibody to ECM Components Treatment % of normalmorhogenesis nonimmune serum 1:10-1:100 79 ± 7.25 (54/68)^(a) Anti-NCl1:10^(b) 0 (0/15) Anti-NCl 1:40 0 (0/6) Anti-NCl 1:80 100 (3/3) Anti-FN1:10^(c) 0 (0/15) Antu-FN 1:50 0 (0/12) Anti-FN 1:100 32 ± 15.5 (10/30)Anti-FN 1:200 33 ± 19.25 (5/15)

[0035] Similar studies were carried out using fibronectin as theinhibitor. Intact fibronectin, proteolytic fragments, RGDS peptides, andantibody to fibronectin were tested to determine their effect on hydracell aggregate development. FIG. 1(B) shows a diagram of therelationship of the various fragments to the intact fibronectinmolecule. Of these, intact fibronectin and its 30-KDa gelatin bindingdomain were found to be effective at 0.5 mg/ml in blocking aggregatedevelopment (FIG. 6). Effectiveness was found to be concentrationdependent. Antibodies against fibronectin also showed blocking effects(Table 1).

[0036] Transmission electron microscopy was used to study theultrastructure of mesoglea under the influences of each exogenouslyintroduced ECM molecule. Blocked aggregates were processed for TEManalysis at various time points of experiments and the ultrastructure oftheir mesoglea was compared to that of control aggregates fixed at thesame time of development. As compared to control group specimens (FIG.5A), the ultrastructure of mesoglea in blocked aggregates was reduced inthickness, irregular at its epithelial border, and appeared to have lostsome of its normal ultrastructural organization (FIG. 5B). Morphometricanalysis (Table 2) indicated that the mesoglea of blocked aggregates wasreduced in thickness by approximately 50% compared to controls and thisreduction was significant as determined using statistical analyses. Thisaltered mesoglea ultrastructure was observed with all blocking reagentstested. TABLE 2 Morphometric Analysis Interepithelial width of mesogleaas Treatment (from tune 0 to 24 hr) reflected by area measurements^(a)BSA^(b) 1434 ± 582  30 kDa^(c) 724 ± 245* Anti-FN^(d) 969 ± 458

[0037] To determine if inhibition of cell proliferation was responsiblefor the blockage of cell aggregate morphogenesis, aggregates weretreated from Time 0 to 96 hr with 10 μM hydroxyurea (HU), which has beenshown to inhibit DNA synthesis in hydra cells (Bode et al., 1976, J.Cell Sci. 20:29-46). Although HU completely blocked DNA synthesis, thedrug did not inhibit morphogenesis of hydra cell aggregates.

[0038] A critical role for collagen in the process of matrix formationfollows from previous pharmacological studies involving lanthryticagents (Barzanski et al., 1974, supra; Sarras et al., 1993, supra). Inthe present demonstration, type IV collagen domains were effective inperturbing mesoglea formation and in blocking aggregate development. Inthis regard, the NC1 domain monomer and the 7S domain fragment were mosteffective in blocking aggregate development when all type IV collagenfragments were compared on an equal molar basis. Others have proposedthat the NC1 domain and the 7S domain are the sites at which the type IVcollagen protomer is involved in intermolecular ridging during formationof the supramolecular network (Martin et al., 1988, Adv. Protein Chem.39:1-50).

[0039] It is not clear why the larger NC1 hexamer and the 80-kDafragment were less effective than the smaller NC1 monomer in blockingdevelopment. The same phenomenon was seen with fibronectin, while themost effective blocker, the 7S fragment, has a mass greater than 100kDa. On a total mass basis, the 7S domain has a relatively highproportion of carbohydrate residues associated with the polypeptidechain (15-18%), and further analysis of these carbohydrate residuesindicate that they have unique structural features such as the presenceof terminal α-D-Gal residues on N-linked oligosaccharide groups(Langenveld et al., 1991, supra; Nayak et al., 1991,. Biol. Chem.26:13978-13987). The inability of type I collagen to block developmentis consistent with the fact that it has not been detected in hydramesoglea.

[0040] The effect of mesoglea components on hydra cell behavior may beviewed at two levels. At one level, mesoglea may be viewed as simply astructural entity which is required as a foundation for stability andmaintenance of the epithelial bilayer. At another level, however, ECMcomponents in mesoglea provide developmental cues which nodule such cellprocesses as cell division, migration, and differentiation during hydramorphogenesis. It is clear that ECM components are not simply structuralmolecules. Fibronectin and type IV collagen have been shown to controlendothelial cell proliferation and differentiation (Tagami et al., 1992,Cell Tissue. Res. 268:225-232: Ingber, 1990, PNAS USA 17:3579-3583). Ithas been shown that the mechanochemical interactions between striatedmuscle cells in jellyfish and grafted mesoglea can induce or inhibit DNAreplication and cell transdifferentiation. In the present examples,while cell proliferation is inhibited in morphologically blockedaggregates, it is also apparent that normal aggregate morphogenesis canoccur even in the absence of cell proliferation. Therefore, a reductionof cell division in blocked aggregates can not account for the blockageobserved in aggregate morphogenesis.

[0041] The homology between the hydra model system and what is knownabout the developmental and regulatory mechanisms of higher organismsmakes the use of type IV collagen fragments in this system applicable tothe same cell and tissue interactions in other organisms. The instantmethods are applicable to, among other things, the inhibition ofmetastasis, control of cell division, reduction of scar tissueformation, intervention in epithelial tissue formation, inhibition ofangiogenesis, reduction of complications due to cell adhesion in organtransplants, inhibition of angiogenic invasion of tissue, or theinhibition of lymphocyte adhesion and mobility.

EXAMPLE 2 Cell Culture Maintenance

[0042] The methods of the instant invention are quite useful for themaintenance of cells in culture, where such maintenance requiresmaintenance of cell phenotype and morphology with minimal adhesion. Itis well recognized that there are many critical factors that contributeto the successful maintenance and propagation of cells in culture.Namely cell division can be anchorage dependent, and in some instanceswill show density-dependent inhibition. Generally most cultures madefrom cells dissociated from tissues, unlike bacteria, are not adapted toliving in suspension and require a solid surface on which to grow anddivide. Cells are now grown in cultures, so that they adhere to plastic.Cells can vary in the requirements of culture media, and the nature ofthe supports, and some cells will not grow without the proper ECMcomponents coated on the plastic dish. Recognized methods for mammaliancell culture can be found in such general references as “Mammalian CellBiotechnology,” edited by M. Butler, Oxford University Press. 1991, and“Readings in Mammalian Cell Culture.” 2nd Edition, edited by R. Pollack,Cold Spring Harbor Laboratory, 1981.

[0043] The methods of the instant invention will provide a means bywhich the requirement for adhesion is eliminated by providing the cellswith an effective amount of the type IV collagen domains which willallow the cells to remain in solution while still appearing to be boundto ECM. This will greatly improve the use of cell cultures inbioreactors and other large scale commercial applications. The use ofNC1 or 7 S domain in about the same effective concentration per cell asdemonstrated in the previous example will keep cultures of cellseffectively bound without adhesion. Further, isolation and manipulationof cells will not require the use of general a pharmacological agentswhich effect many cellular function in an imprecise and non-specificmanner.

[0044] Primary cultures, isolated directly from animal tissues, can beused to form secondary cultures of specific cells. In this manner, thecells can be subcultured for several weeks or months, displaying thephenotype and morphology of the parent cells. Most vertebrate cells willdie in culture after a finite number of divisions, for example humanskin cell's can typically last for several months dividing from 50 to100 times before they die. Variant cell lines can arise however, whichare immortal in that they can be propagated in cell cultureindefinitely. These cells usually grow best when attached to a solidsupport, and typically will cease to grow once they have formed aconfluent layer on the surface, demonstrating contact inhibition. Celllines prepared from cancer cells differ from those prepared from normalcells in many ways. Such cells tend to proliferate without solid supportand will grow to much greater density than normal cells.

[0045] Thus the methods of the instant invention can be used to affectprimary and secondary cell cultures so that they behave as if in contactwith a solid support, but without the effects of cellular contactinhibition. Thus parent cell lines can be maintained in vitro underconditions that will promote the maintenance of cell phenotype andmorphology.

[0046] One area of great interest is the isolation and culture ofembryonic or pluripotent stem cells in vitro for eventual manipulationand use in vivo. The isolation and maintenance of such relativelyundifferentiated cells, and the propagation of such cells would allowfor the production of vast amounts of specific cells which could formthe basis of tissue regeneration or replacement. Thus the teachings ofthe instant invention can be applied to the maintenance of pluripotentcell isolates, and allow for the propagation of these cells whileinhibiting morphological changes and differentiation in vitro.

EXAMPLE 3 In Vitro Effect on Angiogenesis

[0047] Angiogenesis, the process of formation of new blood vessels,plays an important role in physiological processes such as embryonic andpostnatal development as well as in wound repair. Formation of bloodvessels can also be induced by pathological processes involvinginflammation (e.g., diabetic retinopathy and arthritis) or neoplasia(e.g., cancer) (Folkman, 1985, Perspect. Biol. Med., 29, 10).Neovascularization is regulated by angiogenic growth factors secreted bytumor or normal cells as well as the composition of the extracellularmatrix and by the activity of endothelial enzymes (Nicosia andOttinetti, 1990, Lab. Invest., 63, 115).

[0048] During the initial stages of angiogenesis, endothelial cellssprouts appear through gaps in the basement membrane of preexistingblood vessels (Nicosia and Ottinetti, 1990, supra; Schoefl, 1963,Virehous Arch. Pathol. Anat. 337, 97-141; Ausprunk and Folkman, 1977,Microvasc. Res. 14, 53-65; Paku and Paweletz, 1991, Lab. Invest. 63,333-446). As new vessels form, their basement membrane undergoes complexstructural and compositional changes which are believed to affect theangiogenic response (Nicosia et al., 1994, Exp. Biology, 164, 197-206).Early planar culture models have shown that a basement membranemolecules modulate the attachment, migration and proliferation andorganizational behavior of endothelial cells (Nicosia et al., 1994,supra). More recent studies with three-Dimensional aortic culture modelswhich more closely simulate angiogenic conditions that occur duringwound healing in vivo suggest that basement membrane is a dynamicregulator of angiogenesis whose function varies according to itsmolecular components (Nicosia, 1994, supra).

[0049] With modifications, the procedures of Nicosia and Ottinetti(1990), supra, and Nicosia et al (1994), supra, were utilized forexperiments designed to test the effect of Type IV collagen onangiogenesis under in vitro conditions. The model has been used to studythe do of growth factors and extracellular matrix molecules on theangiogenic response and employs aortic rings cultured inthree-dimensional collagen gels under serum-free conditions. Theseexperiments are outlined below.

[0050] A. Methods

[0051] Experiments were performed with Swiss Webster male mice whichwere 1 to 3 months old. Following anesthesia, the thoracic aorta wasexcised under aseptic conditions and transferred to sterile MCDB 131sterile growth medium (Clonctics, San Diego, Calif.) containingantibiotics. Fat was dissected away from the aorta and approximately sixto eight 1 mm thoracic segments were obtained from each specimen.Segments were transferred to 48 well tissue culture plates. The wells ofthese plates were layered with 100 microliters of Matrigel (EHS basementmembrane, Collaborative Biomedical Products, Bedford, Mass.) prior totransfer of the aortic segments. The Matrigel was diluted 1:1 with MCDB131 growth medium prior to use. The segments were centered in the wellsand an additional 100 microliters of Matrigel was then placed over thespecimens. The aortic segments were therefore embedded in the basementmembrane matrix. Each well then received 300 microliters of MCDB 131growth medium. The plates were placed in an incubator maintained at 37°C. with 5% CO₂. Specimens were observed daily over a 7 day period. Newlygrowing microvessels were counted using an inverted phase microscope atvarious times during the culture period, but data is expressed at 3 and5 days of culture. To test for the effect of Type IV collagen onangiogenesis, domains at known concentrations are mixed with theMatrigel and with the MCDB 131 growth medium. Fresh MCDB 131 growthmedium (plus and minus collagen domains) was changed every 3 days.

[0052] B. Results

[0053] After establishing the time course of angiogenesis under controlconditions (Matrigel plus MCDB 131 growth medium), experiments wereperformed using various concentrations of Type IV collagen (isolatedfrom bovine lens) NC1 (hexamer) and 7S domains. Data represents theanalysis of at least 3 specimens per experimental condition. In thefirst experiment (FIG. 7), analysis indicated that a concentration of 50micrograms/ml, NC1 domain and 7S domain significantly inhibitedangiogenesis as monitored at 3 and 5 days of culture. In the secondexperiment, various concentrations of these domains were analyzed. Asindicated in FIG. 8, 7S domain at 50 micrograms/ml again significantlyinhibited angiogenesis at 3 and 5 days. Inhibition was reduced at 5 and0.5 micrograms/ml concentrations. As indicated in FIG. 9, NC1 domain wasless effective in blocking angiogenesis as compared to that observed inthe first experiment (FIG. 7). In addition, as compared to the 7Sdomain, there was less of a correlation between concentration andinhibitory action.

[0054]FIG. 10 are photographs of mouse thoracic aorta segments embeddedin Matrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture in the presence orabsence of 50 ug/ml of Type IV collagen domains. The control specimen(no domains) exhibited growth of microvessels from the cultured tissueinto the matrix (FIG. 10A). In contrast, angiogenesis inhibition wasobserved in tissues cultured in the presence of 50 ug/ml of 7S domain(FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).

[0055] The results of these experiments suggest that as observedpreviously with Hydra cell aggregates, the 7S domain of Type IV collagenis more effective as a blocking agent as compared to the NC1 domain.

[0056] In summary, the present invention is broadly applicable to avariety of in vivo and in vitro uses which include inhibition ofmetastasis, control of cell division, reduction of scar tissueformation, intervention in epithelial tissue formation, inhibition ofangiogenesis, reduction of complications due to cell adhesion in organtransplants, inhibition of angiogenic invasion of tissue, the inhibitionof lymphocyte adhesion and mobility, and the maintenance of pluripotentcell isolates in vitro while inhibiting morphological changes anddifferentiation.

[0057] In practicing the invention, the amount or dosage range of NC1(Hexamer) and 7S domains of Type IV collagen employed is one thateffectively inhibits or disrupts cell adhesion to extracellular matrixcomponents or the formation of functional basal lamina. An inhibitingamount of Type IV collagen domain that can be employed ranges generallybetween about 0.1 and about 500 ug/ml, preferably ranging between about0.5 and about 50 ug/ml.

[0058] While the fundamental novel features of the invention has beenshown and described, it will be understood that various omissions,substitutions and changes in the form and details illustrated may bemade by those skilled in the art without departing from the spirit ofthe invention. It is the intention, therefore, to be limited only asindicated by the scope of the following claims.

What we claim is:
 1. A method for inhibiting basal lamina membraneformation in cell or tissue development comprising contacting the cellor tissue with an effective inhibiting amount of one or more isolateddomains of type IV collagen.
 2. A method for in vitro cultivation ofcells comprising contacting the cells to be cultivated with an effectiveinhibiting amount of an isolated domain of type IV collagen to disruptthe formation of basal lamina membrane or extracellular matrix contacts.3. A method for disrupting basal lamina contact formation with cells ortissues comprising contacting the cells or tissues with an effectiveinhibiting amount of isolated domain of type IV collagen.
 4. A methodfor inhibiting angiogenesis in tissue comprising contacting said tissuewith an effective inhibiting amount of one or more isolated domains oftype IV collagen.
 5. The method of claims 1, 2, 3 or 4, wherein theisolated domain of type IV collagen is the 7S or NC1 domain or proteincontacts having substantially the same structure as the 7S or NC1domain.